Margit M.K. Nass

Mitochondrial DNA: I. Intramitochondrial distribution and structural relations of single- and double-length circular DNA☆

The structure of the DNA of L-cell mitochondria was studied as it may relate to the organelle in vivo. Serial sections of mitochondria revealed two to three DNA-containing regions (nucleoids) per organelle; up to six regions were occasionally observed. Osmotic rupture of freshly isolated mitochondria released 2 to 4% circular DNA dimers (10.1μ, mol. wt 20 × 106) in a population of circular monomers (5.2 μ, mol. wt 10 × 106). The molecules were unbranched, usually folded in half, and either loosely coiled or extended. Most dimers were single circles, either lying free or attached at a polar region to another dimer or two monomers. It appears that each nucleoid may contain one to several monomers and dimers, to yield an average total of two to six molecules per mitochondrion. About 80% of the monomers were associated at polar regions with the mitochondrial membranes; experiments were performed that reduced the possibility of artifact formation. Osmotically shocked mitochondria of ascites tumor cells and adult rat and chicken liver were also compared. The observed membrane associations and the multiplicity of DNA molecules per mitochondrion or per nucleoid may be related to mitochondrial duplication; the high degree of variation in number of molecules per mitochondrion implies redundancy of informational content.

Differential effects of ethidium bromide on mitochondrial and nuclear DNA synthesis in vivo in cultured mammalian cells

Abstract Ethidium bromide (EB), at concentrations of 0.1 to 5 μg/ml, was found to inhibit cell growth and mitochondrial DNA synthesis of cultured L cells (mouse) and BHK cells (hamster), as indicated by cell counts and the incorporation of 3 H-thymidine per μg purified DNA. In contrast, nuclear DNA synthesis of L cells, BHK and polyoma virus transformed BHK cells was actually stimulated to up to 250% of control values by treatment of cells with 0.1 to 2 μg/ml EB for 1 to 2 days. Double-labeling experiments were performed to follow the differential synthesis of mitochondrial and nuclear DNA synthesis and the decay of radioactivity in pre-existing DNA during up to 4 days of treatment with EB. These results and analytical determinations of mitochondrial DNA showed a slightly lower yield of mitochondrial DNA after 4 days of treatment with EB than in controls. The content of protein in mitochondrial fractions was increased to up to 190% of control values. In all three cell lines EB treatment led to a structural alteration of covalently closed mitochondrial DNA, consisting in part of a change to an increased degree of supercoiling and in addition breakage of circular DNA without re-closing. This change involved the preexisting as well as the traces of newly synthesized DNA. These changes were reversible by subsequent growth of cells in EB-free medium.

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells In vivo and in vitro methylation

Abstract Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-3H]methionine; (2) in vivo incorporation of [32P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [3H]deoxycytidine; (4) in vitro methylation of DNAs with 3H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, BHK21 C13 , C13 B4 (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers. Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 106) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells. Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from 3H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.

Studies on mitochondrial tRNA from animal cells: I. A comparison of mitochondrial and cytoplasmic tRNA and aminoacyl-tRNA synthetases

Abstract Mitochondrial and cytoplasmic aminoacyl-tRNAs of rat liver have been compared by chromatography on methylated albumin kieselguhr. This comparison revealed species of leucyl-, tyrosyl-, aspartyl-, valyl- and seryl-tRNA which were found exclusively in mitochondria. These tRNAs did not arise as artifacts during the isolation or acylation procedures. This was shown by the fact that elution profiles could not be altered as a result of denaturing the tRNAs prior to acylation. Furthermore, the relative positions of these tRNAs remained unaltered as a result of interchanging synthetases. It was also found that cytoplasmic synthetases were unable to acylate species of tRNA which were exclusively mitochondrial. These studies add further support to the idea that the protein-synthesizing apparatus of the mitochondria is distinct from that operating in other areas of the cytoplasm.

Mitochondrial DNA. II. Structure and physicochemical properties of isolated DNA.

The molecular topology and proportions of the DNA forms isolated from mitochondria of mouse fibroblasts (L-cells) were studied after removal of contaminating nuclear DNA by DNase. The DNA consisted of 40 to 60% covalently closed circular DNA monomers (4.74 ± 0.02μ; mol.wt 9.1 × 106), including 5% double-length molecules; in addition, up to 10% of the DNA consisted of multiple-length forms of two to four interlocked monomers; the remaining fraction contained mostly open (nicked) circles and some linear fragments; the latter could not be annealed to form circles. The total yield agreed with the chemically determined DNA content of L-cell mitochondria. A 3 to 5% difference in guanine + cytosine content was found for mitochondrial and nuclear DNA by three methods. A comparison of the contour lengths of circular mitochondrial DNA of ascites tumor cells, human liver, chicken liver and rat liver gave values of 4.76 ± 0.04μ, 5.06 ± 0.06μ, 5.20 ± 0.11μ and 4.94 ± 0.09μ, respectively, with slightly higher values for DNA bound to ethidium bromide. The small differences appear to reflect true differences in length, since co-spreading of mixtures of two types of DNA gave bimodal distributions with peak categories representative of the size of each DNA spread individually. The closed circular DNA component of L-cell mitochondria (DNA I) was isolated and studied with respect to the number of tertiary turns and its resistance to denaturation by alkali and heat. By electron microscopy, 33 ± 3 tertiary turns per molecule or 3.6 turns per 1 × 106 molecular weight were found. DNA I sedimented in alkaline CsCl 3.6 to 3.8 times faster than linear DNA. Electron microscopy showed highly twisted monomeric and some dimeric forms in the fast band, monomeric open duplex and single-stranded circles with linear strands peeling off or lying free in the slower bands. The Tm of DNA I in 0.1 SSC (standard saline citrate is 0.15 m-NaCl-0.015 m-sodium citrate) was about 22 °C higher than that of nicked mitochondrial DNA.

Studies on mitochondrial tRNA from animal cells: II. Hybridization of aminoacyl-tRNA from rat liver mitochondria with heavy and light complementary strands of mitochondrial DNA

Abstract The sequence homology of mitochondrial and cytoplasmic aminoacyl-tRNAs with mitochondrial DNA of rat liver has been studied by performing hybridization at low temperatures in the presence of 50% formamide. The tRNA was acylated with radioactive amino acids so that specific aminoacyl-tRNA-DNA hybridization could be followed in the presence of other species of RNA. With a constant amount of DNA and increasing RNA concentrations, a saturation plateau was reached with all tRNAs tested. Competition experiments showed that mitochondrial aminoacyl-tRNA competed far more efficiently with mitochondrial tyrosyl-, phenylalanyl-, seryl- or leucyl-tRNA for hybridization sites on mitochondrial DNA than did cytoplasmic aminoacyl-tRNA. No hybridization was observed with rat liver nuclear DNA or Escherichia coli DNA. Hybridization of mitochondrial aminoacyl-tRNAs was also performed with isolated complementary heavy (H) and light (L) strands of mitochondrial DNA. The DNA was prepared by equilibrium centrifugation in alkaline cesium chloride. It was shown by electron microscopy that at least 20% of the single-stranded DNA molecules present in H and L fractions consisted of full-length 5 μ linear or circular forms. It was found that mitochondrial leucyl- and phenylalanyl-tRNA hybridized exclusively with the H strand, whereas tyrosyl- and seryl-tRNA hybridized exclusively with the L strand of mitochondrial DNA. These studies show (1) that at least four mitochondrial tRNAs are potentially transcribed from mitochondrial DNA; (2) these mitochondrial tRNAs differ in base sequence from their cytoplasmic counterparts; and (3) some species of mitochondrial tRNA may be transcribed in vivo from one strand and others from the complementary strand of mitochondrial DNA.

A novel closed circular duplex DNA in bleached mutant and green strains of Euglena gracilis

Abstract Covalently closed circular duplex DNA, with a contour length of 3.13 ± 0.09 μm, has been isolated by cesium chloride-ethidium bromide equilibrium centrifugation from particulate fractions of permanently bleached mutant and green strains of Euglena gracilis Catenated dimers and oligomers (N = 2 to 6) were also observed. Circles were detected in log or stationary phase cells, dark-grown bleached or ethidium bromide-treated cells. Mitochondria either with or without deoxyribonuclease treatment released linear DNA by osmotic shock. Double labelling experiments showed that the circular DNA had a buoyant density of about 1.701 g·cm−3, which is different from that of nuclear, mitochondrial and chloroplast DNA.

Precise sequence assignment of replication origin in the control region of chick mitochondrial DNA relative to 5' and 3' D-loop ends, secondary structure, DNA synthesis, and protein binding.

The data reported identify for the first time the sequence of an avian mitochondrial heavy-strand replication origin, OH, located only about 12 nucleotides (nt) downstream from the conserved sequence block CSB-1, as well as the sequence of premature synthesis arrest of the 781 (±1) nt D-loop strand, only 6–7 nt downstream from a TAS-like (termination-associated) element. Both sites are associated with putative cruciform secondary structures. A major sequence-specific DNA-binding/cleavage site of a potential regulatory protein, the approximately 36-kDa aMDP1 (shown previously to stimulate mtDNA synthesis), is located about 90 nt upstream of OH. Correlated in vivo analysis of avian genome-length mtDNA replication provides missing evidence on the functional equivalence of D-loop origin with nascent initiation, and on the direction, asymmetry and temporal aspects of a full round of replication. The importance of the results to understanding the regulation of linked replication/transcription and the unusual sequence evolution of avian mtDNA is discussed.

Analysis of the two heavy and light strand origins and the direction of replication of mitochondrial DNA within a detailed physical map of this genome in transformed hamster cells

Abstract The precise physical map positions of the two origins of replication of mtDNA † have been determined in C 13 B 4 cells, a line of Syrian baby hamster kidney fibroblasts transformed with the Bryan strain of Rous sarcoma virus. A detailed physical map with 27 restriction endonuclease recognition sites was constructed, which aligns eight HpaI and two PstI sites with previously mapped cleavage positions for HpaII, HindIII, EcoRI and BamHI. The origin of heavy strand mtDNA replication, which corresponds to the 5′ end of the 7 S initiation sequence in the D-loop structure, was aligned on restriction fragments HpaI A, PstI A, BamHI A, HindIII B, EcoRI B and HpaII F. The location of the origin within the 891 bp fragment HpaII F is approximately 220 bp from the HpaII F A junction which bisects the D-loop. The remaining about 180 bp sequence extends from this border into a ~ 642 bp subfragment of HpaII A, HpaII + EcoRI 8, and expansion of the D-loop proceeds in this direction. Fragment HpaII F can be further cut into at least two subfragments by HaeIII. The identical map position of the D-loop was found for mtDNA from the non-transformed parent cell line BHK 21 C 13 . The 5′ → 3′ orientation of heavy chain replication was specified by the use of exonuclease III. Replication of C 13 B 4 mtDNA appears exclusively asymmetrical, as determined by electron microscopic analysis of replication intermediates and by hybridization of pulse-labeled isolated initiation and expanded sequences with restriction fragments and with H- and L-strands of mtDNA. Unidirectional H-strand displacement synthesis proceeds to 67± 3% of the genome before any duplex synthesis occurs at this location in the opposite direction, marking L-strand initiation. The majority of clustered L-strand initiation sequences flank the restriction site HpaI D B within fragment HpaII E at map position 67.0. The Syrian hamster mtDNA replication system provides an excellent model for studies of genome substructure and function.

Size and configuration of mitochondrial DNA in Euglena gracilis

Abstract 1. (1) The size and structure of mitochondrial DNA from Euglena gracilis (permanently bleached strain) were analyzed by a variety of different isolation and analytical methods. In all cases in which endogenous nuclease activity and shearing of DNA were minimized, small molecules with a mean length of 0.9–1.0 μm ( M r = 1.9 · 10 6 −2.1 · 10 6 ) were found. 2. (2) The DNA was linear under most conditions, but a small percentage of circular molecules ( 1.01 ± 0.05 μ m ) was obtained from mitochondrial fractions, especially after pre-extraction of cells with 80% acetone. The DNA banded at the buoyant density typical of mitochondrial DNA. Membrane-associated DNA molecules which appeared to be circular ( 0.95 ± 0.05 μ m ) and linear forms ( 0.96 ± 0.03 μ m ) were released from osmotically shocked mitochondria. 3. (3) We conclude that Euglena mitochondrial DNA is about 1 μm in length, considerably smaller in size than other known animal and plant mitochondrial DNA. The possibility is discussed that Euglena mitochondrial DNA in situ exists in both circular and linear form.